The goals of this proposal are to determine in breast cancer patients the utilization of a novel signaling pathway emanating from the epidermal growth factor receptor (EGFR) that mediates resistance to drug therapy and to identify the downstream targets of this pathway. Our recent data suggest that ligand-activated EGFR may contribute to drug resistance by translocating to the mitochondria and binding to the mitochondrial enzyme, Cox II (cytochrome c oxidase II). Cox II is a key component of the electron transport chain that binds cytochrome c. It is postulated that binding of EGFR enhances Cox II activity and retention of cytochrome c in the mitochondria, thus reducing drug-induced apoptosis. Cox II binds phospho-Tyr 845 (pY845) of the EGFR, a novel site that is phosphorylated by c-Src following EGF treatment. The adaptor protein, p130Cas (Cas), may play a role in this process by activating c-Src and promoting phosphorylation of Y845 independently of ligand. Mutation of Y845 has no effect on the catalytic activity of the receptor but ablates EGF-induced DNA synthesis and EGF-mediated survival of breast cancer cells following drug treatment. It is not currently known how Cox II binding to pY845 affects Cox II activity or other properties of the mitochondria. The full array of therapeutic agents for which this mechanism applies is also not known. Three approaches will be taken to address these questions. First, we will examine selected breast cancer cell lines that inducibly or transiently express wt or mutant Y845F EGFR for their sensitivity to a panel of drugs that are currently in clinical use. It is expected that cells expressing the wt EGFR will exhibit enhanced resistance, while those expressing the mutant receptor will be more sensitive. Crosstalk between pathways emanating from pY845 and the estrogen receptor (ER) will also be examined in the context of these same cell lines. Second, we will measure alterations in mitochondrial components and functions following drug treatment in cells expressing either the wt or mutant EGFR. Finally, we will explore the possibility that, through its ability to activate c-Src, Cas can promote phosphorylation of Y845 independently of, or in concert with, EGF. In each case, findings from cultured cells and xenograft tumors will be compared to those of human breast cancer samples. Relevance: In spite of significant, recent advances in prevention and treatment of breast cancer, approximately 40,000 women succumb to metastatic or recurrent disease each year. Many fail therapy because their tumors are resistant to the action of cytotoxic or cytostatic drugs. The EGFR family plays an important role in resistance, and we hypothesize that survival signaling through pY845 on the EGFR is a key component of the process. Our studies aim to achieve a better understanding of which cancers demonstrate pY845-dependent resistance to therapy, so that we can target this pathway for intervention. [unreadable] [unreadable] [unreadable] [unreadable]